A vital characteristic of living organisms is their ability to produce biological energy (ATP) efficiently. ATP is required for a myriad of cellular functions, and its synthesis relies on respiratory complexes (RCs) of the electron transport chain (ETC). The Complex III (CIII) is central to ETC, and exists as an individual RC or in association with other RCs to form supercomplexes (SCs). Active SCs that contain CIII together with the Complexes I and IV (respirasomes) are thought to provide stability, channel substrates, and shield toxic intermediates during respiration. Improper functions of RCs and SCs decrease energetic efficiency of cells, and increase toxic reactive oxygen species (ROS), leading to oxidative damages. CIII is a major site of superoxide (O2 ) production on the inter membrane space of mitochondria, and CIII malfunctions lead to .- muscular and neurological diseases. Hence, CIII studies are significant for human health. Our goal is to understand the formation, stability and regulation of CIII containing SCs, their impact on energy transduction, ROS production, and consequences for mitochondrial diseases. Recently, we found a cyt b mutation that renders CIII O2-sensitive, enhances ROS production in both bacteria and humans, and alters SCs organizations in mitochondria. This mutation causes multisystem disorder in humans, and its Rhodobacter capsulatus homologue oxidatively disintegrates the 2Fe2S cofactor of CIII. Using bacterial and human cybrids models, we will investigate similar disease-associated cyt b mutations located at a critical region of cyt b that affects CIII Qo site ROS production and alter SCs organizations. We hypothesize that some of the accessory (i.e., non-catalytic) subunits of RCs act as SC facilitators (SCFs) that might be regulated by O2/ROS to modulate the dynamic plasticity of the ETC. Emerging bacterial SCF candidates are small, single trans membrane-acidic domain (STMD) topology proteins. They include CcoH, FbcQ and DnaJL homologues of mitochondrial Rcf1/HIG2A, bovine CIII subunit VII and MCJ/DNAJ15. Specifically, we will 1- study a group of disease-associated mitochondrial cyt b mutants that produce ROS and alter SCs organizations to elucidate the mechanistic basis of CIII O2 sensitivity and SCs alterations; 2- construct bacterial homologues of the corresponding mitochondrial cyt b mutations, and characterize them for Qo site ROS production and SCs organizations in response to O2/ROS; and 3- determine the role of bacterial homologues of mitochondrial RCs accessory subunits as SCFs, and evaluate their O2 regulation to better understand the physiological roles of SCFs in SCs formation and regulation. We will construct and characterize active fused-SCs, starting with CIII~CIV that we already produced, and evaluate the consequences of perturbing the plasticity of ETC. These studies will contribute to our understanding of the molecular basis of SCs formation, stability and regulation, to establish the interconnections between the CIII Qo site ROS production, SCs alterations, and their disease- associated consequences to eventually improve the diagnostic and treatment of mitochondrial diseases.